RESUMEN
The limitations of TB treatment are the long duration and immune-dampening effects of anti-tuberculosis therapy. The Cell wall plays a crucial role in survival and virulence; hence, enzymes involved in its biosynthesis are good therapeutic targets. Here, we identify Mycobacterium tuberculosis (Mtb) GlmM, (GlmMMtb) engaged in the UDP-GlcNAc synthesis pathway as an essential enzyme. We generated a conditional knockdown strain, Rv-glmMkD using the CRISPR interference-mediated gene silencing approach. Depletion of GlmMMtb affects the morphology and thickness of the cell wall. The Rv-glmMkD strain attenuated Mtb survival in vitro, in the host macrophages (ex vivo), and in a murine mice infection model (in vivo). Results suggest that the depletion of GlmMMtb induces M1 macrophage polarization, prompting a pro-inflammatory cytokine response, apparent from the upregulation of activation markers, including IFNÉ£ and IL-17 that resists the growth of Mtb. These observations provide a rationale for exploring GlmMMtb as a potential therapeutic target.
Asunto(s)
Proteínas Bacterianas , Macrófagos , Mycobacterium tuberculosis , Tuberculosis , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Animales , Ratones , Tuberculosis/inmunología , Tuberculosis/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Femenino , Interacciones Huésped-Patógeno/inmunología , Modelos Animales de Enfermedad , HumanosRESUMEN
Phagosome maturation arrest (PMA) imposed by Mycobacterium tuberculosis ( Mtb ) is a classic tool that helps Mtb evade macrophage anti-bacterial responses. The exclusion of RAB7, a small GTPase, from Mtb -phagosomes underscores PMA. Here we report an unexpected mechanism that triggers crosstalk between the mitochondrial quality control (MQC) and the phagosome maturation pathways that reverses the PMA. CRISPR-mediated p62/SQSTM1 depletion ( p62 KD ) blocks mitophagy flux without impacting mitochondrial quality. In p62 KD cells, Mtb growth and survival are diminished, mainly through witnessing an increasingly oxidative environment and increased lysosomal targeting. The lysosomal targeting of Mtb is facilitated by enhanced TOM20 + mitochondria-derived vesicles (MDVs) biogenesis, a key MQC mechanism. In p62 KD cells, TOM20 + -MDVs biogenesis is MIRO1/MIRO2-dependent and delivered to lysosomes for degradation in a RAB7-dependent manner. Upon infection in p62 KD cells, TOM20 + -MDVs get extensively targeted to Mtb -phagosomes, inadvertently facilitating RAB7 recruitment, PMA reversal and lysosomal targeting of Mtb . Triggering MQC collapse in p62 KD cells further diminishes Mtb survival signifying cooperation between redox- and lysosome-mediated mechanisms. The MQC-anti-bacterial pathway crosstalk could be exploited for host-directed anti-tuberculosis therapies.
RESUMEN
IMPORTANCE: Secreted virulence factors play a critical role in bacterial pathogenesis. Virulence effectors not only help bacteria to overcome the host immune system but also aid in establishing infection. Mtb, which causes tuberculosis in humans, encodes various virulence effectors. Triggers that modulate the secretion of virulence effectors in Mtb are yet to be fully understood. To gain mechanistic insight into the secretion of virulence effectors, we performed high-throughput proteomic studies. With the help of system-level protein-protein interaction network analysis and empirical validations, we unravelled a link between phosphorylation and secretion. Taking the example of the well-known virulence factor of CFP10, we show that the dynamics of CFP10 phosphorylation strongly influenced bacterial virulence and survival ex vivo and in vivo. This study presents the role of phosphorylation in modulating the secretion of virulence factors.
Asunto(s)
Mycobacterium tuberculosis , Humanos , Mycobacterium tuberculosis/metabolismo , Proteínas Bacterianas/metabolismo , Antígenos Bacterianos/metabolismo , Fosforilación , Virulencia , Proteómica , Factores de VirulenciaRESUMEN
Mycobacterium tuberculosis secrets various effector proteins to evade host immune responses for facilitating its intracellular survival. The bacterial genome encodes several unique PE/PPE family proteins, which have been implicated to play important role in mycobacterial pathogenesis. A member of this family, PPE2 have been shown to contain a monopartite nuclear localization signal (NLS) and a DNA binding domain. In this study, we demonstrate that PPE2 protein is present in the sera of mice infected with either M. smegmatis expressing PPE2 or a clinical strain of M. tuberculosis (CDC1551). It was found that exogenously added PPE2 can permeate through the macrophage cell membrane and eventually translocate into the nucleus which requires the presence of NLS which showed considerable homology to HIV-tat like cell permeable peptides. Exogenously added PPE2 could inhibit NO production and decreased mycobacterial survival in macrophages. PPE2-null mutant of M. tuberculosis failed to inhibit NO production and had poor survival in macrophages which could be rescued by complementation with full-length PPE2. PPE2-null mutants also had poor survival in the lungs of infected mice indicating that PPE2 even when present in the bloodstream can confer a survival advantage to mycobacteria.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Animales , Ratones , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Interacciones Huésped-Patógeno , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/metabolismo , Tuberculosis/microbiologíaRESUMEN
Nucleoid-associated proteins (NAPs) regulate multiple cellular processes such as gene expression, virulence, and dormancy throughout bacterial species. NAPs help in the survival and adaptation of Mycobacterium tuberculosis (Mtb) within the host. Fourteen NAPs have been identified in Escherichia coli; however, only seven NAPs are documented in Mtb. Given its complex lifestyle, it is reasonable to assume that Mtb would encode for more NAPs. Using bioinformatics tools and biochemical experiments, we have identified the heparin-binding hemagglutinin (HbhA) protein of Mtb as a novel sequence-independent DNA-binding protein which has previously been characterized as an adhesion molecule required for extrapulmonary dissemination. Deleting the carboxy-terminal domain of HbhA resulted in a complete loss of its DNA-binding activity. Atomic force microscopy showed HbhA-mediated architectural modulations in the DNA, which may play a regulatory role in transcription and genome organization. Our results showed that HbhA colocalizes with the nucleoid region of Mtb. Transcriptomics analyses of a hbhA KO strain revealed that it regulates the expression of â¼36% of total and â¼29% of essential genes. Deletion of hbhA resulted in the upregulation of â¼73% of all differentially expressed genes, belonging to multiple pathways suggesting it to be a global repressor. The results show that HbhA is a nonessential NAP regulating gene expression globally and acting as a plausible transcriptional repressor.
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Proteínas Bacterianas , Hemaglutininas , Mycobacterium tuberculosis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN/química , ADN/metabolismo , Hemaglutininas/genética , Hemaglutininas/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Eliminación de Gen , Proteínas de Unión al ADN/genética , Dominios Proteicos/genética , Microscopía de Fuerza AtómicaRESUMEN
Mycobacterium tuberculosis (Mtb) is a slow-growing, intracellular pathogen that exhibits a high GC-rich genome. Several factors, including the GC content of the genome, influence the evolution of specific codon usage biases in genomes. As a result, the Mtb genome exhibits strong biases for amino acid usage and codon usage. Codon usage of mRNAs affects several aspects of translation, including accuracy, efficiency, and protein folding. Here we address the effect of codon usage biases in determining the translation efficiency of mRNAs in Mtb. Unlike most commonly studied organisms, Mtb carries a single copy of each tRNA gene. However, we show that the relative levels of tRNAs in the Mtb tRNA pool vary by an order of magnitude. Our results show that the codons decoded by the abundant tRNAs indeed show higher adaptability. Moreover, there is a general positive correlation between genomic codon usage and the tRNA adaptability of codons (TAc). We further estimated the optimality of the codon and mRNAs by considering both the TAc and the tRNA demand. These measures did not show any correlation with mRNA abundance and translation efficiency. There was no correlation between tRNA adaptability and ribosome pausing as well. Taken together, we conclude that the translation machinery, and the tRNA pool of an organism, co-evolve with the codon usage to optimize the translation efficiency of an organism. Thus the deleterious effect of maladapted codons is not pronounced.
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Mycobacterium tuberculosis , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Biosíntesis de Proteínas/genética , Codón/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismoRESUMEN
SigA (σA) is an essential protein and the primary sigma factor in Mycobacterium tuberculosis (Mtb). However, due to the absence of genetic tools, our understanding of the role and regulation of σA activity and its molecular attributes that help modulate Mtb survival is scant. Here, we generated a conditional gene replacement of σA in Mtb and showed that its depletion results in a severe survival defect in vitro, ex vivo, and in vivo in a murine infection model. Our RNA-seq analysis suggests that σA either directly or indirectly regulates â¼57% of the Mtb transcriptome, including â¼28% of essential genes. Surprisingly, we note that despite having â¼64% similarity with σA, overexpression of the primary-like σ factor SigB (σB) fails to compensate for the absence of σA, suggesting minimal functional redundancy. RNA-seq analysis of the Mtb σB deletion mutant revealed that 433 genes are regulated by σB, of which 283 overlap with the σA transcriptome. Additionally, surface plasmon resonance, in vitro transcription, and functional complementation experiments reveal that σA residues between 132-179 that are disordered and missing from all experimentally determined σA-RNAP structural models are imperative for σA function. Moreover, phosphorylation of σA in the intrinsically disordered N-terminal region plays a regulatory role in modulating its activity. Collectively, these observations and analysis provide a rationale for the centrality of σA for the survival and pathogenicity of this bacillus.
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Proteínas Bacterianas , Viabilidad Microbiana , Mycobacterium tuberculosis , Factor sigma , Factor sigma/genética , Factor sigma/metabolismo , Animales , Ratones , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Transcriptoma , Tuberculosis/microbiología , Eliminación de Secuencia , Viabilidad Microbiana/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/genéticaRESUMEN
The emergence of drug resistance in Mycobacterium tuberculosis (Mtb) is alarming and demands in-depth knowledge for timely diagnosis. We performed genome-wide association analysis using 2237 clinical strains of Mtb to identify novel genetic factors that evoke drug resistance. In addition to the known direct targets, we identified for the first time, a strong association between mutations in DNA repair genes and the multidrug-resistant phenotype. To evaluate the impact of variants identified in the clinical samples in the evolution of drug resistance, we utilized knockouts and complemented strains in Mycobacterium smegmatis and Mtb. Results show that variant mutations compromised the functions of MutY and UvrB. MutY variant showed enhanced survival compared with wild-type (Rv) when the Mtb strains were subjected to multiple rounds of ex vivo antibiotic stress. In an in vivo guinea pig infection model, the MutY variant outcompeted the wild-type strain. We show that novel variant mutations in the DNA repair genes collectively compromise their functions and contribute to better survival under antibiotic/host stress conditions.
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Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Animales , Cobayas , Antituberculosos/farmacología , Estudio de Asociación del Genoma Completo , Farmacorresistencia Bacteriana Múltiple/genética , Reparación del ADN , Mutación , Pruebas de Sensibilidad Microbiana , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
We describe steps for gDNA isolation from mycobacterium strains isolated from guinea pig lungs. We detail steps for infection of guinea pigs with Mycobacterium tuberculosis, followed by in vitro growth, gDNA isolation, and whole genome sequencing. We also describe an ex vivo competition experiment to determine the selective advantage of one strain over another. We include details for WGS and mutation spectrum analysis. The protocol can be used to identify mutations that arise in other pathogenic bacteria. For complete details on the use and execution of this protocol, please refer to Naz et al. (2021).
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Mycobacterium tuberculosis , Tuberculosis , Cobayas , Animales , Mycobacterium tuberculosis/genética , Tuberculosis/microbiología , Pulmón/microbiología , Secuenciación Completa del GenomaRESUMEN
The unique structural components of cell membranes of Gram-positive bacteria, Gram-negative bacteria, and mycobacteria provide an excellent therapeutic target for developing highly specific antimicrobials. Here, we report the synthesis of nine cholic acid (CA)-derived amphiphiles, where three hydroxyl groups of CA were tethered to dimethylamino pyridine and the C24-carboxyl group was conjugated with different alkyl chains. Structure-activity investigations revealed that amphiphile 1 harboring a methyl group has antimicrobial activity against mycobacterial species. On the other hand, amphiphile 7 containing an octyl chain was selective against Gram-positive and Gram-negative bacilli. Biochemical assays confirmed the selective membrane permeabilization abilities of amphiphiles 1 and 7. Importantly, we demonstrate the selective actions of amphiphiles in clearing biofilms, intracellular bacteria, and wound infections. Therefore, for the first time, we show that the unique structural features of CA-derived amphiphiles dictate selective activity against specific bacterial species.
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Antibacterianos , Bacterias Grampositivas , Ácido Cólico/farmacología , Antibacterianos/farmacología , Antibacterianos/química , Bacterias Gramnegativas , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Mycobacterium tuberculosis encodes ~200 transcription factors that modulate gene expression under different microenvironments in the host. Even though high-throughput chromatin immunoprecipitation sequencing and transcriptome sequencing (RNA-seq) studies have identified the regulatory network for ~80% of transcription factors, many transcription factors remain uncharacterized. EmbR is one such transcription factor whose in vivo regulon and biological function are yet to be elucidated. Previous in vitro studies suggested that phosphorylation of EmbR by PknH upregulates the embCAB operon. Using a gene replacement mutant of embR, we investigated its role in modulating cellular morphology, antibiotic resistance, and survival in the host. Contrary to the prevailing hypothesis, under normal growth conditions, EmbR is neither phosphorylated nor impacted by ethambutol resistance through the regulation of the embCAB operon. The embR deletion mutant displayed attenuated M. tuberculosis survival in vivo. RNA-seq analysis suggested that EmbR regulates operons involved in the secretion pathway, lipid metabolism, virulence, and hypoxia, including well-known hypoxia-inducible genes devS and hspX. Lipidome analysis revealed that EmbR modulates levels of all lysophospholipids, several phospholipids, and M. tuberculosis-specific lipids, which is more pronounced under hypoxic conditions. We found that the EmbR mutant is hypersusceptible to hypoxic stress, and RNA sequencing performed under hypoxic conditions indicated that EmbR majorly regulates genes involved in response to acidic pH, hypoxia, and fatty acid metabolism. We observed condition-specific phosphorylation of EmbR, which contributes to EmbR-mediated transcription of several essential genes, ensuring enhanced survival. Collectively, the study establishes EmbR as a key modulator of hypoxic response that facilitates mycobacterial survival in the host. IMPORTANCE Mycobacterium tuberculosis modulates its transcriptional machinery in response to dynamic microenvironments encountered within the host. In this study, we identified that EmbR, a transcription factor, plays important roles in modulating cellular morphology, antibiotic resistance, and survival in the host. We found that EmbR undergoes condition-specific phosphorylation for its activation. Together, the study establishes a key role of EmbR as a transcriptional activator of genes belonging to multiple pathways, viz., virulence, secretion, or polyketide synthesis, that aid in mycobacterial survival during hypoxia and within the host.
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Proteínas Bacterianas , Mycobacterium tuberculosis , Factores de Transcripción , Factores de Virulencia , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Hipoxia , Mycobacterium tuberculosis/metabolismo , Factores de Transcripción/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Applying antibiotics to susceptible bacterial cultures generates a minor population of persisters that remain susceptible to antibiotics but can endure them for extended periods. Recent reports suggest that antibiotic persisters (APs) of mycobacteria experience oxidative stress and develop resistance upon treatment with lethal doses of ciprofloxacin or rifampicin. However, the mechanisms driving the de novo emergence of resistance remained unclear. Here, we show that mycobacterial APs activate the SOS response, resulting in the upregulation of the error-prone DNA polymerase DnaE2. The sustained expression of dnaE2 in APs led to mutagenesis across the genome and resulted in the rapid evolution of resistance to antibiotics. Inhibition of RecA by suramin, an anti-Trypanosoma drug, reduced the rate of conversion of persisters to resistors in a diverse group of bacteria. Our study highlights suramin's novel application as a broad-spectrum agent in combating the development of drug resistance.
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Farmacorresistencia Bacteriana , Mycobacterium tuberculosis , Antibacterianos/farmacología , Ciprofloxacina/farmacología , Farmacorresistencia Bacteriana/genética , Mycobacterium tuberculosis/genética , Rifampin/farmacologíaRESUMEN
Tuberculosis (TB), including extrapulmonary TB, is responsible for more than one million deaths in a year worldwide. Existing methods of mycobacteria detection have poor sensitivity, selectivity, and specificity, especially in human tissues. Herein, the synthesis of a cholic acid-derived fluorescent probe (P4) that can specifically stain the mycobacterium species is presented. It is shown that P4 probe specifically binds with mycobacterial lipids, trehalose monomycolate, and phosphatidylinositol mannoside 6. P4 probe can detect mycobacteria in polymicrobial planktonic cultures and biofilms with high specificity, selectivity, and sensitivity. Moreover, it can detect a single mycobacterium in the presence of 10 000 other bacilli. Unlike the probes that depend on active mycobacterial enzymes, the membrane-specific P4 probe can detect mycobacteria even in formalin-fixed paraffin-embedded mice and human tissue sections. Therefore, the ability of the P4 probe to detect mycobacteria in different biological milieu makes it a potential candidate for diagnostic and prognostic applications in clinical settings.
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Mycobacterium tuberculosis , Tuberculosis , Animales , Colorantes Fluorescentes , Humanos , Ratones , Adhesión en Parafina , Sensibilidad y Especificidad , Tuberculosis/diagnósticoRESUMEN
Delhi, the national capital of India, experienced multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreaks in 2020 and reached population seropositivity of >50% by 2021. During April 2021, the city became overwhelmed by COVID-19 cases and fatalities, as a new variant, B.1.617.2 (Delta), replaced B.1.1.7 (Alpha). A Bayesian model explains the growth advantage of Delta through a combination of increased transmissibility and reduced sensitivity to immune responses generated against earlier variants (median estimates: 1.5-fold greater transmissibility and 20% reduction in sensitivity). Seropositivity of an employee and family cohort increased from 42% to 87.5% between March and July 2021, with 27% reinfections, as judged by increased antibody concentration after a previous decline. The likely high transmissibility and partial evasion of immunity by the Delta variant contributed to an overwhelming surge in Delhi.
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COVID-19/epidemiología , COVID-19/virología , Genoma Viral , Adolescente , Adulto , COVID-19/inmunología , COVID-19/transmisión , Niño , Humanos , Evasión Inmune , India/epidemiología , Epidemiología Molecular , Filogenia , Reinfección , Estudios Seroepidemiológicos , Adulto JovenRESUMEN
We present a non-immunogenic, injectable, low molecular weight, amphiphilic hydrogel-based drug delivery system (TB-Gel) that can entrap a cocktail of four front-line antitubercular drugs, isoniazid, rifampicin, pyrazinamide, and ethambutol. We showed that TB-Gel is more effective than oral delivery of the combination of four drugs in reducing the mycobacterial infection in mice. Results show that half the dose of chemotherapeutic drugs is sufficient to achieve a comparable therapeutic effect to that of oral delivery.
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Antituberculosos , Hidrogeles , Animales , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Etambutol , Isoniazida , Ratones , PirazinamidaRESUMEN
Mycobacterium tuberculosis (Mtb) has evolved diverse cellular processes in response to the multiple stresses it encounters within the infected host. We explored available TnSeq datasets to identify transcription factors (TFs) that are essential for Mtb survival inside the host. The analysis identified a single TF, Rv1332 (AosR), conserved across actinomycetes with a so-far uncharacterized function. AosR mitigates phagocyte-derived oxidative and nitrosative stress, thus promoting mycobacterial growth in the murine lungs and spleen. Oxidative stress induces formation of a single intrasubunit disulphide bond in AosR, which in turn facilitates AosR interaction with an extracytoplasmic-function sigma factor, SigH. This leads to the specific upregulation of the CysM-dependent non-canonical cysteine biosynthesis pathway through an auxiliary intragenic stress-responsive promoter, an axis critical in detoxifying host-derived oxidative and nitrosative radicals. Failure to upregulate AosR-dependent cysteine biosynthesis during the redox stress causes differential expression of 6% of Mtb genes. Our study shows that the AosR-SigH pathway is critical for detoxifying host-derived oxidative and nitrosative radicals to enhance Mtb survival in the hostile intracellular environment.
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Actinobacteria/genética , Homeostasis/genética , Mycobacterium tuberculosis/genética , Factores de Transcripción/genética , Animales , Proteínas Bacterianas/genética , Femenino , Regulación Bacteriana de la Expresión Génica/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Oxidación-Reducción , Estrés Oxidativo/genética , Regiones Promotoras Genéticas/genética , Factor sigma/genética , Transcripción Genética/genéticaRESUMEN
N-acetyl glucosamine-1-phosphate uridyltransferase (GlmU) is a bifunctional enzyme involved in the biosynthesis of Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a critical precursor for the synthesis of peptidoglycan and other cell wall components. The absence of a homolog in eukaryotes makes GlmU an attractive target for therapeutic intervention. Mycobacterium tuberculosis GlmU (GlmUMt) has features, such as a C-terminal extension, that are not present in GlmUorthologs from other bacteria. Here, we set out to determine the uniqueness of GlmUMt by performing in vivo complementation experiments using RvΔglmU mutant. We find that any deletion of the carboxy-terminal extension region of GlmUMt abolishes its ability to complement the function of GlmUMt. Results show orthologs of GlmU, including its closest ortholog, from Mycobacterium smegmatis, cannot complement the function of GlmUMt. Furthermore, the co-expression of GlmUMt domain deletion mutants with either acetyl or uridyltransferase activities failed to rescue the function. However, co-expression of GlmUMt point mutants with either acetyl or uridyltransferase activities successfully restored the biological function of GlmUMt, likely due to the formation of heterotrimers. Based on the interactome experiments, we speculate that GlmUMt participates in unique interactions essential for its in vivo function.
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Acetiltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Complejos Multienzimáticos/metabolismo , Mutación , Mycobacterium tuberculosis/crecimiento & desarrollo , Tuberculosis/microbiología , UDP-Glucosa-Hexosa-1-Fosfato Uridiltransferasa/metabolismo , Acetiltransferasas/química , Acetiltransferasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Conformación Proteica , Dominios Proteicos , UDP-Glucosa-Hexosa-1-Fosfato Uridiltransferasa/química , UDP-Glucosa-Hexosa-1-Fosfato Uridiltransferasa/genéticaRESUMEN
Serine/threonine-protein kinases in Mycobacterium tuberculosis (Mtb) form a preeminent regulatory system required to establish and maintain the infection in the host. Herein, we sought to decipher the biological role of PknL with the help of a gene replacement mutant RvΔpknL. Deletion of pknL results in the compromised growth under redox stress. The mutant showed significant survival defects in peritoneal macrophages, a significant decrease in the ability to establish infections and disseminate to the spleen in the murine model of infection. While the absence of pknL has no impact on either MIC or CFUs of ciprofloxacin and rifampicin treated bacilli, it increases the survival ~1.5-2.5 log fold upon isoniazid or ethambutol treatment. Collectively, data suggests that PknL aids in combating stress conditions in vitro, ex vivo, and in vivo and reduces the efficacy of isoniazid and ethambutol.
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Antituberculosos/farmacología , Etambutol/farmacología , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Animales , Proteínas Bacterianas/genética , Eliminación de Gen , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genéticaRESUMEN
Tuberculosis caused by Mycobacterium tuberculosis (Mtb) is a significant public health concern, exacerbated by the emergence of drug-resistant TB. To combat the host's dynamic environment, Mtb encodes multiple DNA repair enzymes that play a critical role in maintaining genomic integrity. Mtb possesses a GC-rich genome, rendering it highly susceptible to cytosine deaminations, resulting in the occurrence of uracils in the DNA. UDGs encoded by ung and udgB initiate the repair; hence we investigated the biological impact of deleting UDGs in the adaptation of pathogen. We generated gene replacement mutants of uracil DNA glycosylases, individually (RvΔung, RvΔudgB) or together (RvΔdKO). The double KO mutant, RvΔdKO exhibited remarkably higher spontaneous mutation rate, in the presence of antibiotics. Interestingly, RvΔdKO showed higher survival rates in guinea pigs and accumulated large number of SNPs as revealed by whole-genome sequence analysis. Competition assays revealed the superior fitness of RvΔdKO over Rv, both in ex vivo and in vivo conditions. We propose that compromised DNA repair results in the accumulation of mutations, and a subset of these drives adaptation in the host. Importantly, this property allowed us to utilize RvΔdKO for the facile identification of drug targets.